The funnel landscape model predicts that protein folding proceeds through multiple kinetic pathways. Experimental evidence is presented for more than one such pathway in the folding dynamics of a globular protein, cytochrome c. After photodissociation of CO from the partially denatured ferrous protein, fast time-resolved CD spectroscopy shows a submillisecond folding process that is complete in Ϸ10 ؊6 s, concomitant with heme binding of a methionine residue. Kinetic modeling of time-resolved magnetic circular dichroism data further provides strong evidence that a 50-s heme-histidine binding process proceeds in parallel with the faster pathway, implying that Met and His binding occur in different conformational ensembles of the protein, i.e., along respective ultrafast (microseconds) and fast (milliseconds) folding pathways. This kinetic heterogeneity appears to be intrinsic to the diffusional nature of early folding dynamics on the energy landscape, as opposed to the late-time heterogeneity associated with nonnative heme ligation and proline isomers in cytochrome c.
Protein unfolding during guanidine HCl denaturant titration of the reduced and oxidized forms of cytochrome c is monitored with magnetic circular dichroism (MCD), natural CD, and absorption of the heme bands and far‐UV CD of the amide bands. Direct MCD spectral evidence is presented for bis‐histidinyl heme ligation in the unfolded states of both the reduced and oxidized protein. For both redox states, the unfolding midpoints measured with MCD, which is an indicator of tertiary structure, are significantly lower than those measured with far‐UV CD, an indicator of secondary structure. The disparate titration curves are interpreted in terms of a compound mechanism for denaturant‐induced folding and unfolding involving a molten globulelike intermediate state (MG) with near‐native secondary structure and nonnative tertiary structure and heme ligation. A comparison of the dependence of the free energy of formation of the MG intermediate on the redox state with the known contributions from heme ligation and solvation suggests that the heme is significantly more accessible to solvent in the MG intermediate than it is in the native state. © 2000 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 57: 29–36, 2000
Time-resolved circular dichroism measurements, over a spectral range from 300 to 700 nm, were made at delays of 5, 100 and 500 μs after room temperature photoexcitation of bovine rhodopsin in lauryl maltoside suspension. The purpose was to provide more structural information about intermediate states in the activation of rhodopsin and other G protein-coupled receptors. In particular, information was sought about photointermediates that are isochromic or nearly isochromic in their unpolarized absorbance. The circular dichroism spectrum of lumirhodopsin, obtained after correcting the 5 μs difference CD data for the rhodopsin bleached, was in reasonable agreement with the lumirhodopsin CD spectrum obtained previously by thermal trapping at -76°C. Similarly, the metarhodopsin II spectrum obtained at 500 μs delay was also in agreement with the results of previous work on the temperature trapped form of metarhodopsin II. However, the CD of the mixture formed at 100 μs delay after photoexcitation, whose only visible absorbing component is lumirhodopsin, could not be accounted for near 480 nm in terms of the initially formed, 5 μs lumirhodopsin CD spectrum. Thus, the CD spectrum of lumirhodopsin changes on the time scale from 5 to 100 μs, showing reduced rotational strength in its visible band, possibly associated with either a process responsible for a small spectral shift that occurs in the lumirhodopsin absorbance spectrum at earlier times or the Schiff base deprotonation-reprotonation which occurs during equilibration of lumirhodopsin with the Meta I 380 photointermediate. Either explanation suggests a chromophore conformation change closely associated with deprotonation which could be the earliest direct trigger of activation. The x-ray crystal structure of rhodopsin provides a fundamental basis for any model of visual pigment activation. However, the x-ray structure of rhodopsin, while rich in 3D structural information lacks a time coordinate, making that picture just the beginning of a yet to be constructed motion picture. Based on the initial coordinates, a detailed series of activation steps remains to be elucidated, and those cannot be adapted from another system, since a heptahelical membrane bound protein triggered by isomerization of its N-retinylidene Schiff base chromophore is essentially unprecedented. What is needed to fill in the activation mechanism blanks are dynamic measurements, especially those with structural content, conducted at early enough times that they can be interpreted in terms of small perturbations of the initial crystal structure data.Traditionally, UV/visible absorbance measurements have the highest time resolution of any technique by several orders of magnitude. As a light activated protein, rhodopsin exposes more of its activation steps for optical study than do most protein systems. Given the wide time range accessible to UV/visible absorbance measurement, it is not surprising that they reveal a complicated series of intermediate steps in visual pigment activation (see Sch...
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